Alexander Fleming (1881-1955)

74

               Proceedings of the Royal Society of Medicine

4

Wolff, by methods which need not be described here, succeeded in preparing pure
lysozyme from egg-white. He obtained about 0.1 mg. per c.c. of egg-white. The
purified lysozyme was an amorphous light yellow substance easily soluble in water.
It has the same antibacterial properties as the original egg-white.

Methods of demonstrating lysozyme action.—For the effective and dramatic
demonstration of bacteriolysis by lysozyme it is necessary to have a sensitive test
organism. I obtained a coccus (which for convenience I named M. lysodeikticus)
which is very sensitive to lysozyme action. This sensitive coccus was used to work
out the properties and distribution of lysozyme in 1921 and 1922. It has been
subcultured at intervals on agar for ten years and its properties are apparently
unchanged. The use of this sensitive coccus has, however, had an unfortunate effect
in that it has given the impression that lysozyme is only active on a small number
of non-pathogenic organisms and is really of little practical importance. To this
point I will return later.

Bacteriolysis of this sensitive test-coccus by lysozyme is certainly the most
dramatic method of bacteriolysis I have ever seen. In experiments with immune
bacteriolytic serum one is accustomed to mix the serum in high concentration with
a relatively small number of bacteria and incubate for some time before a definite
result is seen. Even then more often than not the bacterial suspension is not
completely dissolved although the bacteria may be largely broken up. With
lysozyme, on the other hand, the bacteriolytic action is rapid, clearing of the opaque
suspension is complete, and the lytic action may be seen even with very high
dilutions. For instance, lysis manifest to the naked eye can be obtained with
human tears diluted 1 in 2,000,000 and with egg-white of half that strength.

If to 1 c.c. of an opaque suspension of the test coccus, 10 c.mm. of human tears
are added at a temperature of 50° C., there is obvious clearing of the suspension within
thirty seconds, and in about two minutes the opacity will have completely
disappeared. Such rapid and complete lysis was quite strange to me until I worked
with lysozyme.

Another striking demonstration can be made by taking a well-grown plate culture
of M. lysodeikticus from the incubator and immediately placing on it, while it is still
warm, a drop of human tears or hen’s egg-white and a control drop of saline. The
plate is then tipped up so that the drops run down the plate in streaks. Within
thirty seconds the culture under the tear streak will become less opaque, and in a
few minutes the opaque growth will be replaced by a yellowish, gelatinous, perfectly
transparent sheet. No change occurs along the control saline streak.

The inhibitory and bacteriolytic action of lysozyme can well be shown by
punching out a disc from the centre of the agar plate and filling this in with agar
containing a lysozyme-containing material (tears, egg-white, or a piece of tissue).
The whole plate is then flooded with agar containing M. lysodeikticus. On
incubation for twenty-four hours there is copious growth of M. lysodeikticus except
for a distance of perhaps a centimetre around the “ lysozyme well.” After two days
the centre portion of the growth will have become transparent, and on longer
cultivation this lytic change gradually spreads as the lysozyme diffuses throughout
the agar until, after a week or more, it reaches the margin of the plate (see fig. 2).

Formation of resistant strains.—Fleming and Allison (1922) showed that when
secretions or tissues were embedded in a plate seeded with M. lysodeikticus there
was first a zone of complete inhibition around, but after a longer or shorter interval
there might appear in this clear zone isolated colonies of M. lysodeikticus. When
these were subcultured and titrated with lysozyme they were found to be markedly
more resistant than was the original culture. This resistance persisted in subse-
quent generations and we found that such resistant strains, which had only once been
exposed to lysozyme, maintained their resistance for at least eight months, in spite of
repeated subculture.